Abstract Our TSRI-ARC has focused on preclinical work on the cellular, neurochemical, and molecular mechanisms of alcohol dependence, with a major interest in the role of the central nucleus of the amygdala (CeA) in excessive alcohol drinking. However, there is a great need to understand the mechanisms that mediate dependence- induced drinking motivated directly by withdrawal/abstinence symptoms. The Neurophysiology Project will continue to study the neuroadaptations induced by alcohol dependence in the CeA, and will now investigate upstream neuronal circuits that are responsible for dysregulation of the CeA during alcohol abstinence. Corticotropin-releasing factor (CRF) and CRF1 receptors are involved in the ethanol-induced increase in GABA release in the CeA and the CRF system is upregulated after ethanol dependence. Chronic CRF1 antagonism blocked alcohol dependence-induced increases in ethanol consumption. Notably, the transition to alcohol dependence also dysregulates executive function, and the infralimbic (IL) subdivision of the mPFC exerts “top- down” control over the amygdala to regulate emotional aspects of goal-directed behaviors. Thus, this renewal application focuses on the overall hypothesis that ethanol dependence and withdrawal are driven by the recruitment of CRF and serotonin (5-HT) signaling in cortical-amygdala circuits. We will study neural function during ethanol abstinence and characterize how long those neuroadaptive changes persist. In particular, our goal is to characterize neuroadaptations in the CRF and 5-HT systems and their effects in the CeA and IL through common cellular systems to induce a maladaptation in neural function that promotes ethanol withdrawal-induced anxiety-like behavior. Our project is designed to test the hypothesis that dependence and withdrawal (early withdrawal = 2-8 h;; late withdrawal = 2 weeks) differentially alter responses to 5-HT and CRF in CeA neurons (Specific Aim 1) and will affect the excitability of IL pyramidal neurons in layer V that project to the CeA (Specific Aim 2). Finally, Specific Aim 3 will electrophysiologically assess the ethanol-induced synaptic and molecular mechanisms of candidate drugs that are identified and tested in preclinical animal models (Contet and Animal Models Core) and subsequently tested in the clinical component (Mason). The project will use IL and CeA brain slices and standard whole-cell patch-clamp and cell-attached electrophysiological methods, as well as measures of protein levels and chemogenetic and behavioral testing that will involve continued collaboration with the George/Zorrilla, Contet, Martin-Fardon, and Mason projects and Animal Models Core. It is imperative to provide data that elucidate the cellular basis of the susceptibility of a...